Human and mammalian stem cell-derived neuron survival polypeptides

ABSTRACT

The present invention relates to human, rat and mouse stem cell-derived neuron survival factor polypeptides (SDNSF), a process for producing them, cDNA encoding SDNSF, a vector comprising the cDNA, host cells transformed by the vector, an antibody against SDNSF, pharmaceutical compositions containing SDNSF or the antibody, a method of assaying SDNSF, a reagent for assaying SDNSF, and a screening method using SDNSF. The polypeptides are effective in the survival of nerve cells and, therefore, efficacious in treating injury to the central nerve system caused by brain infarction, brain hemorrhage, spinal cord injury, etc.

TECHNICAL FIELD

[0001] The present invention relates to human, rat and mouse Stemcell-Derived Neuron Survival Factors (hereinafter, simply referred to as“SDNSF”). In more detail, it relates to human, rat and mouse SDNSF, aprocess for producing them, cDNA encoding SDNSF, a vector comprising thecDNA, host cells transformed by the vector, an antibody against SDNSF, apharmaceutical compositions containing SDNSF or the antibody, a methodof assaying SDNSF, a reagent for assaying SDNSF, and a screening methodusing SDNSF.

BACKGROUND ART

[0002] In adult brain, it is a long established theory that a new neuronis not generated, and that in an injury to the central nerve systemcaused by brain infarction, brain hemorrhage, spinal cord injury, etc.and in a neurodegenerative disease such as Parkinson's disease andamyotrophic lateral sclerosis (ALS), recovery of the function which themovement was lost according to the cell death of a neuron was difficult.In recent years, however, it was shown that a neuron was newly generatedin adult brain (hippocampus, cerebral cortex association area, lateralcerebral ventricle) of higher mammals such as human and monkey, and thata new neuron in these regions was generated from a neuronal stem cell.It was also demonstrated that a neuronal stem cell existed in agedpeople's brain and it could differentiate into a neuron. These factssuggest that cerebral regenerative medical treatment is not limited tocell therapy which transplants cells, but therapy which activatesinherent neuronal stem cells directly by adminstrating a drug medicinecontaining protein or compound or by gene therapy thchnology ispossible.

[0003] In order to obtain a specific polypeptide or a cDNA encoding it,there have been generally employed methods comprising confirming thetarget biological activity in a tissue or a cell culture medium and thencloning of a gene through the isolation and purification of apolypeptide and further methods comprising expression-cloning of a genewith the guidance of the biological activity. However, it is frequentlyobserved that a gene, which has been cloned with the guidance of acertain activity, codes for a known polypeptide since manyphysiologically active polypeptides occurring in vivo have variousbiological activities. Further, most intravital physiologically activefactors are generated only in a trace amount or under a specificphysiological condition, which makes the isolation and purificationthereof and the confirmation of biological activity difficult.

DISCLOSURE OF THE INVENTION

[0004] The inventors focused on novel factors (polypeptides), especiallysecretory proteins and membrane proteins which have secretion signals,useful for the medical treatment or diagnosis of an injury to thecentral nerve system or a neurodegenerative disease, diagnosis orresearch of a brain tumor, and examined repeatedly and wholeheartedly.Consequently, the inventors have isolated the novel polypeptide moleculeconcerning a neuronal stem cell, and found out that it is available forregenerative medical treatment above-mentioned and for specific markerof a neuron.

[0005] The present inventors have studied the cloning of genes forproliferation and differentiation factors in hemetopoietic and immunesystems. They have paid attention to the fact that most secretoryproteins such as proliferation and/or differentiation factors (forexample various cytokines) and membrane proteins such as receptorsthereof (hereinafter these proteins will be referred to generally assecretory proteins and the like) have sequences called signal peptidesin the N-termini. Extensive studies have been conducted to provide aprocess for efficiently and selectively cloning genes encoding signalpeptides. As a result, a process (signal sequence trap (SST) method) hasbeen devised using animal cells whereby the existence of a signalpeptide can be easily examined (Japanese Patent No. 2,879,303).Furthermore, a process (the yeast SST method) for massively and easilyisolating genes encoding signal peptides by using yeast was alsodeveloped based on the same concept (U.S. Pat. No. 5,536,637).

[0006] By using this method, the inventors have identified successfullya novel secretory protein which is generated by neural stem cellsderived from adult rat hippocampus and a cDNA encoding the protein, andfound out a full-length cDNA from neural stem cells derived from adultrat hippocampus based on the information.

[0007] The inventors have confirmed that the polypeptide had survivalsupporting activity to some cerebral neurons (primary cultured cellsfrom hippocampus nerve and stem cells derived from hippocampus), asmight be explained in detail behind, and completed the invention. Thepolypeptide is the useful factor as which the function was specified.

[0008] The cDNA sequence which this invention offers was identified as arat SDNSF clone shown in SEQ ID No.1 or 2, and isolated from cDNAlibrary prepared from stem cells derived from hippocampus based on theinformation obtained by yeast SST method. The rat SDNSF clone shown inSEQ ID No.1 is a full-length cDNA encoding the secretory protein (it isindicated as rat SDNSF protein in the invention).

[0009] The cDNA sequence which this invention offers was identified as ahuman SDNSF clone shown in SEQ ID No.5 or 6, and isolated from cDNAlibrary prepared from stem cells derived from hippocampus based on theinformation obtained by yeast SST method. The human SDNSF clone shown inSEQ ID No.5 is a full-length cDNA encoding the secretory protein (it isindicated as human SDNSF protein in the invention).

[0010] The cDNA sequence which this invention offers was identified as amouse SDNSF clone shown in SEQ ID No.9 or 10, and isolated from cDNAlibrary prepared from stem cells derived from hippocampus based on theinformation obtained by yeast SST method. The mouse SDNSF clone shown inSEQ ID No.9 is a full-length cDNA encoding the secretory protein (it isindicated as mouse SDNSF protein in the invention).

[0011] The nucleotide sequence encoding rat SDNSF was compared withsequences in GenBank and NCBI utilizing the programs BLASTN, FASTA andUNIGENE, and the amino acid sequence of rat SDNSF was compared withsequences utilizing the programs BLASTP, Fly Database and SwissProt, toreveal no identical sequence. From the results, it became clear that thepolypeptide of the invention was a novel secretory protein.

[0012] From the facts that the polypeptide of the invention hassustentation activity on neuron in a portion of brain, has EF handmotifs in spite of secretory protein, is a possible cytokine which isregulated by extracellular calcium or calcium from organelle involved insecretory pathway, and has no sequence homology to known neurotrophins,it is thought that the polypeptide is a cytokine which promotesformation or support of survival of neurons other than sympatheticnerve, sensory nerve, neuron in spinal cord motor nerve nucleus andcholinergic nerve in basal ganglia which are known to be neurotrophindependence by the analysis using deficit mouse of known neurotrophingene, and that the polypeptide leads to elucidate etiologies ofneurodegenerative diseases and to treat them.

[0013] The present invension relates to:

[0014] (1) A substantially purified form of a polypeptide comprising theamino acid sequence shown in SEQ ID NO. 4, 8 or 12, a homologue thereof,a fragment thereof or a homologue of the fragment,

[0015] (2) The polypeptide according to (1) that comprises the aminoacid sequence shown in SEQ ID NO. 4, 8 or 12,

[0016] (3) A cDNA encoding the polypeptide according to (1) or (2),

[0017] (4) The cDNA according to (3), comprising the nucleotide sequence shown in SEQ ID NO. 1, 2, 5, 6, 9 or 10, or a fragm entselectively hybridized to the sequence,

[0018] (5) A replication or expression vector comprising the cDNAaccording to (3) or (4),

[0019] (6) A host cell transformed with the replication or expres sionvector according to (5),

[0020] (7) A process for producing the polypeptide according to (1) or(2), which comprises culturing the host cell accord ing to (6) under acondition effective to express the polyp eptide according to (1) or (2),

[0021] (8) A monoclonal or polyclonal antibody against the polype ptideaccording to (1) or (2),

[0022] (9) A pharmaceutical composition containing the polypeptid eaccording to (1) or (2) or the antibody according to (8), in associationwith a pharmaceutically acceptable excipie nt and/or carrier,

[0023] (10) A pharmaceutical composition effective for the medica ltreatment of a neurodegenerative disease, containing the polypeptideaccording to (1) or (2) or the antibody accord ing to (8), inassociation with a pharmaceutically accepta ble excipient and/orcarrier,

[0024] (11) The pharmaceutical composition according to (10), in whichthe neurodegenerative disease is an injury to the central nerve systemby brain infarction, brain hemorrhage, spinal cord injury, etc.,

[0025] (12) A method for measuring the polypeptide according to (1) or(2),

[0026] (13) An immunochemical method for measuring the polypeptideaccording to (1) or (2), comprising using the antibody according to (8),

[0027] (14) A reagent for detecting the polypeptide according to (1) or(2), comprising using the method according to (12) or (13),

[0028] (15) A reagent for testing tumor, comprising using the methodaccording to (12) or (13),

[0029] (16) The reagent according to (14), in which the tumor is a braintumor,

[0030] (17) A method for screening a compound having agonistic orantagonistic activity against the polypeptide, comprising using thepolypeptide according to (1) or (2),

[0031] (18) An agent for the treatment of an injury to the central nervesystem which comprises, as an active ingredient, the polypeptideaccording to (1) or (2),

[0032] (19) The reagent according to (19), in which the injury to thecentral nerve system is the one caused by a brain infarction,

[0033] (20) The reagent according to (19), in which the injury to thecentral nerve system is the one caused by a brain hemorrhage, and

[0034] (21) The reagent according to (19), in which the injury to thecentral nerve system is the one caused by a spinal cord injury.

DETAILED DESCRIPTION

[0035] A polypeptide of SEQ ID NO. 4, 8 or 12 in substantially purifiedform will generally comprise the polypeptide in a preparation in whichmore than 90%, eg. 95%, 98% or 99% of the polypeptide in the preparationis that of the SEQ ID NO. 4, 8 or 12.

[0036] A polypeptide homologue of the SEQ ID NO. 4, 8 or 12 will be atleast 70%, preferably at least 80% or 90%, and more preferably 95%homologous to the polypeptide over a region of at least 20, preferablyat least 30 for instance 40, 60, 80 or 100 contiguous amino acids. Suchpolypeptide homologues will be referred to below as a polypeptideaccording to the invention.

[0037] Furthermore, fragments of SEQ ID NO. 4, 8 or 12 or its homologueswill be at least 10, preferably at least 15 for example 20, 25, 30, 40,50 or 60 amino acids in length of the polypeptide.

[0038] A cDNA capable of selectively hybridizing to the cDNA of SEQ IDNO. 1, 2, 5, 6, 9 or 10 will be generally at least 70%, preferably atleast 80% or 90%, and more preferably at least 95% homologous to thecDNA of SEQ ID NO. 1, 2, 5, 6, 9 or 10 over a region of at least 20,preferably at least 30, for instance 40, 60, 80 or 100 contiguousnucleotides. Such cDNA will be encompassed by the term “cDNA accordingto the invention”.

[0039] Fragments of SEQ ID NO. 1, 2, 5, 6, 9 or 10 will be at least 10,preferably at least 15 for example 20, 25, 30 or 40 nucleotides inlength of the cDNA. Such fragments will be encompassed by the term “cDNAaccording to the invention”.

[0040] Because SDNSF protein of the invention is secreted in greatquantities from undifferentiated neuroblastoma and glioblastoma amongbrain tumors, but not secreted from differentiated glioma, and there isno available neural tumor marker secreted in circulating blood or spinalfluid, SDNSF protein could be the first marker for undifferentiatedneural tumor of which detection is possible in blood.

[0041] A further embodiment of the invention provides replication andexpression vectors comprising cDNA according to the invention. Thevectors may be, for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promotor for the expressionof the said DNA and optionally a regulator of the promoter. The vectormay contain one or more selectable marker genes, for example anampicillin resistance gene. The vector may be used in vitro, for examplefor the production of RNA corresponding to the cDNA, or used totransform a host cell.

[0042] A further embodiment of the invention provides host cellstransformed or transfected with the vectors for the replication andexpression of cDNA according to the invention, including the cDNA of SEQID NO. 1, 2, 5, 6, 9 or 10 or the open reading frame thereof. The cellswill be chosen to be compatible with the vector and may for example bebacterial, yeast, insect or mammalian.

[0043] A further embodiment of the invention provides a method ofproducing a polypeptide which comprises culturing host cells of thepresent invention under conditions effective to express a polypeptide ofthe invention. Preferably, cultivation is carried out under conditionsin which the polypeptide of the invention is expressed and thengenerated from the host cells.

[0044] A cDNA according to the invention may also be inserted into thevectors described above in an antisense orientation in order to providefor the production of antisense RNA. Such antisense RNA maybe used in amethod of controlling the levels of a polypeptide of the invention in acell.

[0045] An embodiment of the invention also provides monoclonal orpolyclonal antibodies against a polypeptide of the present invention. Afurther embodiment of the invention provides a process for production ofmonoclonal or polyclonal antibodies to the polypeptides of the presentinvention. Monoclonal antibodies maybe prepared by common hybridomatechnology using polypeptides of the present invention or fragmentsthereof as an immunogen. Polyclonal antibodies may also be prepared bycommon means which comprises inoculating host animals, for example a rator a rabbit, with polypeptides of the invention and recovering immuneserum.

[0046] An embodiment of the invention also provides pharmaceuticalcompositions containing a polypeptide of the present invention, or anantibody against the polypeptide, in association with a pharmaceuticallyacceptable excipient and/or carrier.

[0047] (A) As the polypeptide of the present invention referred to above1, those which have deficiency in a part of their amino acid sequence(e.g., a mature polypeptide consisted of the only essential sequence forrevealing a biological activity in an amino acid sequence shown in SEQID NO. 4), those which have a part of their amino acid sequence replacedby other amino acids (e.g., those replaced by an amino acid having asimilar property) and those which have other amino acids added orinserted into a part of their amino acid sequence, as well as thosecomprising the amino acid sequence shown in SEQ ID NO. 4, 8 or 12.

[0048] As known well, there are one to six kinds of codon encoding oneamino acid (for example, one kind of codon for Methionine, and six Kindsof codon for leucine are known). Accordingly, the nucleotide sequence ofcDNA can be changed without changing the amino acid sequence of thepolypeptide.

[0049] (B) The cDNA of the present invention referred to above 3includes every group of nucleotide sequences encoding polypeptides ofSEQ ID NO. 4, 8 or 12 shown in (A). There is a probability that yield ofa polypeptide is improved by changing a nucleotide sequence.

[0050] (C) The cDNA specified in SEQ ID NO. 2, 6 or 10 is an embodimentof the cDNA shown in (B), and indicates the sequence of natural form.

[0051] (D) The cDNA shown in SEQ ID NO. 1, 5 or 9 indicates the sequencein which natural non-coding region is added to the cDNA specified in(C).

[0052] A cDNA carrying nucleotide sequence shown in SEQ ID NO. 1, 5 or 9is prepared by the following method.

[0053] First, Yeast SST method (see U.S. Pat. No. 5,536,637) is brieflydescribed below.

[0054] Yeast such as Saccharomyces cerevisiae should secrete invertaseinto the medium in order to take sucrose or raffinose as a source ofenergy or carbon (Invertase is an enzyme to cleave raffinose intosucrose and melibiose, sucrose into fructose and glucose.). It is knownthat many of known mammalian signal sequences make yeast secrete itsinvertase.

[0055] From this knowledge, SST method was developed as a screeningmethod to find a novel signal peptide which enables invertase secretionof yeast from mammalian cDNA library with growth of the yeast onraffinose medium as an index.

[0056] Non-secretory type invertase gene SUC2 (GENBANK Accession No.V01311) lacking initiation codon ATG was inserted to yeast expressionvector to prepare yeast SST vector pSUC2. In this expression vector, ADHpromoter, ADH terminator (both were derived from AAH5 plasmid (Gammerer,Methods in Enzymol. 101, 192-201, 1983)), 2μ ori (as a yeast replicationorigin), TRP1 (as a yeast selective marker), ColE1 ori (as a E. Colireplication origin) and ampicillin resistance gene (as a drug resistancemarker) were inserted. Mammalian cDNA was inserted into the upstream ofSUC2 gene to prepare yeast SST cDNA library. Yeast lacking secretorytype invertase, was transformed with this library.

[0057] If inserted mammalian cDNA encodes a signal peptide, the yeastcould survive on raffinose medium as a result of restoring secretion ofinvertase. By culturing yeast in colonies to prepare plasmids anddetermine the nucleotide sequence of the insert cDNAs, it is possible toidentify novel signal peptide rapidly and easily.

[0058] Preparation of yeast SST cDNA library is as follows:

[0059] (1) mRNA is isolated from targeted cells, a double-strand cDNA issynthesized by using random primer with certain restriction enzyme(enzyme I) recognition site,

[0060] (2) the double-strand cDNA is ligated to adapter containingcertain restriction endonuclease (enzyme II) recognition site differentfrom enzyme I, digested with enzyme I and fractionated in a appropriatesize,

[0061] (3) the obtained cDNA fragment is inserted into yeast expressionvector on the upstream region of invertase gene of which signal peptideis deleted and the library is transformed.

[0062] Detailed description of each step is as follows:

[0063] In step (1), mRNA is isolated from mammalian organs or cell linesafter stimulating them with appropriate stimulator if necessary by knownmethods (as described in Molecular Cloning (Sambrook, J., Fritsch, E. F.and Maniatis, T., Cold Spring Harbor Laboratory Press, 1989) or CurrentProtocol in Molecular Biology (F. M. Ausubel et al, John Wiley & Sons,Inc.) unless otherwise specified). A suitable tissue may be heart offetal mouse. Double-strand cDNA synthesis using random primer isperformed by known methods.

[0064] Any sites may be used as restriction endonuclease recognitionsite I which is linked to adapter and restriction endonucleaserecognition site II which is used in step (2), insofar as both sites aredifferent each other. Preferably, XhoI is used as enzyme I and EcOR1 asenzyme II.

[0065] In step (2), ends of cDNA are blunted with T4 DNA polymerase, andligated to enzyme II adapter and digested with enzyme I. Fragment cDNAis analyzed with agarose-gel electrophoresis (AGE) and cDNA fractionranging in size from 300 to 800 bp is selected. As mentioned above, anyenzyme may be Used a's enzyme II insofar as it is not same with theenzyme I.

[0066] In step (3), cDNA fragment obtained in step (2) is inserted intoyeast expression vector on the upstream region of invertase gene ofwhich signal peptide is deleted. E. coli is transformed with theexpression vector. Many vectors are known as yeast expression plasmidvector. For example, YEp24 is also functioned in E. Coli. PreferablypSUC2 as described above is used.

[0067] Many host E. Coli strains are known as usable for transformation,preferably DH10B competent cell is used. Any known transformation methodis available, preferably it is performed by electropolation method.Transformant is cultured by conventional methods to obtain cDNA libraryfor yeast SST method.

[0068] However, not all of the cloned cDNA fragments are introduced intothis cDNA library. Further, not all of the gene fragments encode unknown(novel) signal peptides. It is therefore necessary to screen a genefragment encoding for an unknown signal peptide from the library.

[0069] Therefore, screening of fragments containing a sequence encodingan appropriate signal peptide is performed by transformation of the cDNAlibrary into Saccharomyces cerevisiae (e.g. YT455 strain) lacking theinvertase gene or strain which artificially lack the gene (it may beprepared by known methods.). Transformation of yeast is performed byknown methods, e.g. lithium acetate method. Transformant is cultured ina selective medium, then transferred to a medium containing raffinose asa carbon source. Survival colonies are selected and then plasmid iscollected. Survival colonies on a raffinose-medium indicates that somesignal peptide of secretory protein was inserted to this clone.

[0070] With respect to isolated positive clones, the nucleotide isdetermined. As to a cDNA encoding unknown protein, full-length clone maybe isolated by using cDNA fragment as a probe, and then the full-lengthnucleotide sequence is determined. The manipulation is performed byknown methods.

[0071] Once the nucleotide sequences shown in SEQ ID NO. 1, 5 or 9 aredetermined partially or preferably fully, it is possible to obtain cDNAencoding mammalian protein itself, homologue or subset. By screeningcDNA library or mRNA derived from mammals by PCR method with anysynthesized oligonucleotide primers or by hybridization with anyfragment as a probe, it is possible to obtain cDNA encoding othermammalian homologue protein from other mammalian cDNA or genome library.

[0072] If the cDNA obtained above contains a nucleotide sequence of cDNAfragment obtained by SST (or homologous sequence thereof), it impliesthat the cDNA encodes signal peptide. Accordingly, it is clear that thelength of the cDNA is full or almost full. (All signal sequences existat N-termini of a protein and are encoded at 5′-termini of open readingframe of cDNA.)

[0073] By known methods, the confirmation of full-length may be carriedout by Northern analysis with the said cDNA as a probe. The cDNA isassumed to have almost complete length if the length of the cDNA isalmost the same with the length of the mRNA obtained in the hybridizingband.

[0074] The present invention provides both types of protein, i.e.,full-length and mature. The full-length proteins are specified with theamino acid sequences translated from the nucleotides shown in SEQ IDNO.4, 8 or 12. The mature proteins are obtained by expression insuitable mammal cells or other host cells transformed by the full-lengthDNA shown in SEQ ID NO.1, 5 or 9. Sequences of mature proteins could bepredicted from full-length amino acid sequences.

[0075] Once the nucleotide sequences shown in SEQ ID No. 1, 2, 5, 6, 9or 10 are determined, cDNAs of the present invention are obtained bychemical synthesis, or by hybridization making use of nucleotidefragments which are chemically synthesized as a probe. Furthermore,cDNAs of the invention are obtained in desired amount by transforming avector that contains the DNA into a proper host, and culturing thetransformant.

[0076] The polypeptides of the present invention may be prepared by:

[0077] (1) isolating and purifying from an organism or a cultured cell,

[0078] (2) chemically synthesizing, or

[0079] (3) using recombinant DNA technology, preferably, by the methoddescribed in (3) in an industrial production.

[0080] Examples of expression system (host-vector system) for producinga polypeptide by using recombinant DNA technology are the expressionsystems of bacteria, yeast, insect cells and mammalian cells.

[0081] In the expression of the polypeptide, for example, in E. Coli,the expression vector is prepared by adding the initiation codon (ATG)to 5′ end of a cDNA encoding mature peptide, connecting the cDNA thusobtained to the downstream of a proper promoter (e.g., trp promoter, lacpromoter, λ PL promoter, and T7 promoter), and then inserting it into avector (e.g., pBR322, pUC18 and pUC19) which functions in an E. colistrain.

[0082] Then, an E. coli strain (e.g., E. coli DH1 strain, E. coli JM109strain and E. coli HB101 strain) which is transformed with theexpression vector described above may be cultured in an appropriatemedium to obtain the desired polypeptide. When a signal peptide ofbacteria (e.g., signal peptide of pelB) is utilized, the desiredpolypeptide may be also released in periplasm. Furthermore, a fusionprotein with other polypeptide may also be produced.

[0083] In the expression of the polypeptide, for example, in mammaliancells, for example, the expression vector is prepared by inserting theDNA encoding nucleotide shown in SEQ ID NO. 1, 2, 5, 6, 9 or 10 into thedownstream of a proper promoter (e.g., SV40 promoter, LTR promoter andmetallothionein promoter) in a proper vector (e.g., retrovirus vector,papilloma virus vector, vaccinia virus vector and SV40 vector). A propermammalian cell (e.g., monkey COS-1 cell, COS-7 cell, Chinese hamster CHOcell, mouse L cell etc.) is transformed with the expression vector thusobtained, and then the transformant is cultured in a proper medium, thesecretory protein of the present invention can be secreted into theculture medium as the aimed polypeptide. Then, by linking to cDNAfragment coding other polypeptides, for example, common region(Fcportion) of antibody, fusion proteins can be produced. Polypeptidesobtained by the method above can be isolated and purified byconventional biochemical methods.

INDUSTRIAL APPLICABILITY

[0084] A polypeptide and cDNA encoding it of the invention are thoughtto have one or more effects or biological activities (The effects orbiological activities relevant to assay enumerated below are included).

[0085] Administration or use of the protein or of cDNA coding theprotein (for example, gene therapy (including regenerative therapy) orvectors suitable for cDNA transfection) may provide the effect orbiological activities described about the protein of the invention.

[0086] The polypeptide of the invention has sustentation activity onneuron in a portion of brain (primary cultured cells from hippocampusnerve and stem cells derived from hippocampus) and, therefore, isefficacious in treating neurodegenerative disease (injury in the centralnerve system by brain infarction, brain hemorrhage, spinal cord injury,etc.).

[0087] Quantitative analysis of the polypeptide of the present inventionin vivo can be performed using polyclonal or monoclonal antibodiesagainst the polypeptide of the present invention. It can be used instudies on relationship between this polypeptide and disease, ordiagnosis of disease, etc. The polyclonal and the monoclonal antibodiescan be prepared using this polypeptide or its fragment as an antigen byconventional methods.

[0088] Identification, purification or gene cloning of known or unknownproteins (ligands) which are connected with the polypeptide of thepresent invention can be performed using the polypeptide of the presentinvention by, for example, preparation of the affinity-column.

[0089] Identification of molecules which interact with the polypeptide,molecular cloning of the gene may be conducted, for example, bywest-western blot, using the polypeptide, or by yeast two-hybrid method,using the cDNA (desirably cDNA coding the polypeptide).

[0090] Screening, which can identify agonists or antagonists against thepolypeptide receptor and inhibitors against interaction betweenreceptors and signal transduction molecule's can be performed by usingthe polypeptide.

[0091] For example, the screening could be performed by the followingsteps:

[0092] (a) The polypeptide of the invention, compound to be screened andreaction mixture including cells are mixed (the reaction mixtureincludes markers which are transferred into cells as the cell grows andpeptides except for the polypeptide for efficient observation of thefunction of the polypeptide.) under condition which the cells arenormally stimulated by the polypeptide, then,

[0093] (b) It is determined whether the compound is a useful agonist orantagonist by measuring the cell growth.

[0094] The cDNA of the invention may be useful not only as an importantand essential template in production of the polypeptide of the presentinvention which is expected to have a considerable utility, but also fordiagnoses and treatments of hereditary diseases (treatments of genedeficiency or treatments which anti-sense DNA(RNA)s intercept expressionof polypeptides, etc). In addition, genomic DNAs may be isolated byusing cDNA of the invention as a probe.

[0095] For the usage for above mentioned diseases, administration of thepolypeptide of the invention or the antibodies against the polypeptideof the invention can be carried out in systemic or local, generallyperoral or parenteral ways. Oral, intravenous andintracerebroventricular administration are preferred.

[0096] The dosage to be administered depends upon age, body weight,symptom, desired therapeutic effect, route of administration, andduration of the treatment etc. In human adults, one dose per person isgenerally between 100 μg and 100 mg by oral administration up to severaltimes per day, or between 10 μg and 100 mg by parenteral administrationup to several times per day.

[0097] As mentioned above, the doses to be used depend upon variousconditions. Therefore, there are cases in which doses lower than orgreater than the ranges specified above may be used.

[0098] The compounds of the present invention may be administered assolid compositions, liquid compositions or other compositions for oraladministration, or as injections, liniments or suppositories etc. forparenteral administration.

[0099] Examples of solid compositions for oral administration includecompressed tablets, pills, capsules, dispersible powders and granulesetc. Examples of capsules include hard capsules and soft capsules.

[0100] In such solid compositions, one or more of the active compound(s)is or are admixed with at least one inert diluent (such as lactose,mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose,starch, polyvinylpyrrolidone, magnesium metasilicate aluminate, etc.).The compositions may also comprise, as is normal practice, additionalsubstances other than inert diluents: e.g. lubricating agents (such asmagnesium stearate etc.), disintegrating agents (such as cellulosecalcium glycolate, etc.), stabilizing agents (such as human serumalbumin, lactose etc.), and assisting agents for dissolving (such asarginine, asparaginic acid etc.).

[0101] The tablets or pills may, if desired, be coated with a film ofgastric or enteric materials (such as sugar, gelatin, hydroxypropylcellulose or hydroxypropylmethyl cellulose phthalate, etc.), or becoated with more than two films. And then, coating may includecontainment within capsules of absorbable materials such as gelatin.

[0102] Liquid compositions for oral administration may containpharmaceutically-acceptable emulsions, solutions, suspensions, syrupsand elixirs, and also may contain inert diluent(s) commonly used(purified water, ethanol etc.). Besides inert diluents, suchcompositions may also comprise adjuvants (such as wetting agents,suspending agents, etc.), sweetening agents, flavoring agents, perfumingagents, and preserving agents.

[0103] Other compositions for oral administration include spraycompositions which may be prepared by known methods and which compriseone or more of the active compound(s). Spray compositions may compriseadditional substances other than inert diluents: e.g. stabilizing agentssuch as sodium sulfite etc., stabilizing agents providing for isotonicbehavior, isotonic buffer (sodium chloride, sodium citrate, citric acid,etc.). For preparation of such spray compositions, for example, themethods described in the U.S. Pat. Nos. 2,868,691 and 3,095,355 may beused.

[0104] Injections for parenteral administration include sterile aqueousor non-aqueous solutions, suspensions and emulsions. In aqueous ornon-aqueous solutions or suspensions, one or more active compound(s) isor are admixed with at least one inert diluent (s). Aqueous diluents maybe distilled water for injection, physiological salt solution, etc.Inert non-aqueous diluents (s) maybe propylene glycol, polyethyleneglycol, oil of the plant such as olive oil, alcohol such as ethanol,POLYSOLBATE 80™, etc.

[0105] Such compositions may comprise additional preserving agents,wetting agents, emulsifying agents, dispersing agents, stabilizing agent(such as human serum albumin, lactose, etc.), and assisting agents suchas assisting agents for dissolving (arginine, asparaginic acid, etc.)

BRIEF DESCRIPTION OF THE DRAWINGS

[0106]FIG. 1(A) shows an alignment of deduced amino acid sequences ofhuman, mouse, and rat SDNSF, (B) existence of two EF hand motifs(calsium binding motif) in the downstream of the signal peptide,

[0107]FIG. 2 shows the EF hand motifs of SDNSF and those of calmodulin,calcium secretory protein, have common sequences,

[0108]FIG. 3 is the result of Western Blot analysis with anti-SDNSFantibody and anti-FLAG antibody, showing SDNSF protein is secreted intothe culture medium,

[0109]FIG. 4 is the result of blotting with the cDNA fragment³²P-labeled and purified by gel as a probe,

[0110]FIG. 5 shows the expression of SDNSF mRNA in primary neurons,astrocytes, neural stem cells (embryo NSCs, adult NSCs undifferentiatedand adult NSCs differentiated), rat glioma C6, mouse neuroblast N18 andhuman glioblastoma UG251,

[0111]FIG. 6 shows the expression of SDNSF protein in primaryastrocytes, primary neurons, adult NSCs, mouse neuroblast N18 and humanglioblastoma UG251,

[0112]FIG. 7(A) (B) shows the effect of SDNSF addition on the survivalof hippocampal neurons and neural stem cells,

[0113]FIG. 8 shows that SDNSF improves hippocampal neurons viavility ina dose dependent manner (Significant differences versus control areindicated by an asterisk (*) (P<0.01)),

[0114]FIG. 9 shows the effect of SDNSF on the survival of neural stemcells cultured in the absence of FGF-2 (Significant differences versuscontrol are indicated by an asterisk (*) (P<0.01)),

[0115]FIG. 10 shows the effect of SDNSF on self-renewal of neural stemcells by counting neurospheres formed in the absence of FGF-2(Significant differences versus control are indicated by an asterisk (*)(P<0.01)),

[0116]FIG. 11 shows the effect of SDNSF on the differentiation of neuralstem cells into neurons in the neurospheres formed in the presence ofSDNSF without FGF-2 (Significant differences between SDNSF-treated,FGF-2-treated and control groups are indicated by an asterisk (*)(P<0.01)).

BEST MODE FOR CARRYING OUT THE INVENTION

[0117] The present invention is more specifically explained by means ofthe following examples regarding SDNSF, but is not limited only to theseexamples.

EXAMPLE 1 Preparation of poly(A)+ RNA

[0118] Total RNA was extracted from PZ5 cells, which was cloned fromneural stem cells derived from adult rat hippocampus, with TRI_(zol)reagent™ (purchased from Life Technologies, Inc), and poly(A)⁺ RNA waspurified with Oligotex-dT30<Super>™ (purchased from Roche).

EXAMPLE 2 Construction of Yeast SST cDNA Library

[0119] Double-stranded cDNA was synthesized from above-describedpoly(A)⁺ RNA with a primer, in which XhoI site 9 mer was connected with,

[0120] 5′-TCC CGA TTG AAT TCT AGA CCT GCC TCG AGN NNN NNN NN-3′ (SEQ IDNO.13)

[0121] by using Super Script Choice System™ (purchased from LifeTechnologies, Inc). It was connected with EcORI adaptor (purchased fromGIBCOERL) by using DNA ligation kit Ver.2™ (purchased from TAKARA SYUZO,hereinafter this kit was used for ligation of cDNA), digested with XhoI,electrophoresed in agarote gel and 400-800 bp cDNAs were cut off fromthe gel. The cDNAs were inserted into EcOR1/XhoI site of pSuc2t7lori(see U.S. Pat. No. 5,536,637), introduced into E. coli DH10 byelectropolation to obtain cDNA library for yeast SST method.

EXAMPLE 3 Screening by SST and Sequencing of Positive Clones

[0122] Plasmids were prepared from the cDNA library, yeast YTK12 wastransformed with the plasmids by lithium acetate method (see CurrentProtocols in Molecular Biology 13.7.1) and plated onto selection mediumfor yeast transformants (CMO-Trp medium) lacking tryptophan. After 48hours incubation at 30° C., colonies (transformants) were replicatedonto YPR plates, of which carbon sourse is raffinose, by using AccutranReplica Plater ™ (purchased from Schleicher & Schuell) and incubated for14 days at 30° C.

[0123] On day 3 or later, each yeast colony was purified by streakingonto YPR plates and incubated for 48 hours at 30° C. Single colony wasinoculated into YPD medium and incubated for further 48 hours at 30° C.,then plasmid was prepared. PCR reaction was performed with two kinds ofprimers having the sequences at the ends of pSUC2 cloning site (primerfor sense-strand is biotin-labelled) by known method to amplify insertcDNA, biotin-labelled single-stranded cDNA was purified using Dynabeads™(purchased from DYNAL) and then sequenced by cycle-sequencing methodusing fluorescence-dye terminator with DNA Sequencing kit (DyeTerminator Cycle Sequencing Ready Reaction™) (purchased from AppliedBiosystems Inc.). DNA sequencer 373 (Applied Biosystems Inc.) was usedfor reading the nucleotide sequence (hereinafter sequencing was carriedout by this method).

[0124] DNA sequences thus obtained and deduced amino acid sequences werecompared with sequences in data bases, it became clear that a clonenamed SDNSF was a novel cDNA. Therefore, full-length cDNA cloning wastried with this fragment cDNA of SDNSF clone (hereinafter referred to as“SDNSF SST fragment cDNA”). A comparison of the deduced amino acidsequence with those of known signal peptides indicated that SDNSF SSTfragment cDNA had signal peptide both functionally and structurally.

EXAMPLE 4 Full-Length cDNA Cloning and Sequencing

[0125] One million plaques obtained from PZ5 cDNA library weretransferred to nylon membrane. Hybridization was carried out with³²P-labelled rat SDNSF SST fragment cDNA as a probe by known method toobtain many positive clones. One clone among them was isolated,introduced into E. coli DH5α, and its plasmid was prepared. Aftersequencing of 5′ region of the insert and confirming the DNA containedthe sequence of rat SDNSF SST fragment cDNA, full length sequencing wasperformed to obtain the sequence shown in SEQ ID No.1. The open readingframe was also determined to obtain the translated amino acid sequenceshown in SEQ ID No.2 and the deduced amino acid sequence shown in SEQ IDNo.4.

[0126] The amino acid sequence and nucleotide sequence encoding thepolypeptide of the invention (referred to as rat SDNSF polypeptide) werecompared with sequences in NCBI data base to reveal no identicalsequence. Furthermore, it became clear that rat SDNSF polypeptide had notransmembrane region and that rat SDNSF polypeptide was a novelsecretory protein.

[0127] The result of motif search revealed that SDNSF had a signalpeptide and two EF hand motifs (calcium binding motif) downstream of thesignal peptide (FIG. 1). EF hand motif of SDNSF and that of calmodulin,a calcium secretory protein, have common sequences (FIG. 2). It is rarethat a secretory protein has the EF hand motif, however, it is reportedthat BM-40 and its related proteins have a similar structure. The EFhands in BM-40 is suspected to be involed in the conformation change ofthe protein depending on the concentration of calcium in vesicle andsecretion efficiency of BM-40 (Literature 1: Busch E et. al., Calciumaffinity cooperativity and domain interaction of extracellular EF-handspresent in BM-40., J. Biol. Chem., 275(33), 25508-15(2000))

EXAMPLE 5 Sequencing of Human and Mouse SDNSF Genes

[0128] Homology searches on mammalian ESTs and UNIGENE DNA data basesrevealed human and mouse ESTs homologous to rat SDNSF.

[0129] Consequently, the inventors isolated full length human and mouseSDNSF genes using the sequence information by known method and sequencedcompletely to obtain the nucleotide sequence's shown in SEQ ID No.5 and9, respectively. The open reading frame was also determined to obtainthe deduced amino acid sequence shown in SEQ ID No.8 and 12,respectively. It was revealed from above information that said human andmouse clones were full length and their amino acid sequences were 87%and 90% identical to that of rat SDNSF, respectively.

[0130] The nucleotide sequences and amino acid sequences of the humanand mouse SDNSF were compared with sequences on nucleotide and aminoacid data bases to reveal no identical sequence as was the case with ratSDNSF. From this, it became clear that the polypeptides of the inventionwere novel secretory proteins as well.

EXAMPLE 6 Homology Searches on Data Bases for Non-Mammals

[0131] Homology searches on nematode and drosophila data bases revealedthat F55A11.1, which had been reported to be a virtual protein ofnematode, and CG12817, which had been reported to be a gene product ofdrosophila, had 20 to 30% identities in amino acid sequence with thepolypeptides. These data base searches suggest that SDNSF genes arehighly conserved.

EXAMPLE 7 Preparation of Anti-SDNSF Polyclonal Antibody

[0132] Three kinds of rat SDNSF partial polypeptides were synthesized bysolid-phase method and conjugated to Keyhole limpet hemocyanin (KLH),

[0133] Asp Lys Ser Thr Val His Asp Gln Glu His Ile Met Glu His Leu GluCys-KLH

[0134] (amino acid sequence 15-30 in SEQ ID No.4)

[0135] His Lys Glu Glu Gly Ser Glu Gln Val Pro Pro Met Ser Glu Asp GluCys-KLH

[0136] (amino acid sequence 74-89 in SEQ ID No.4)

[0137] KLH-Cys Asp Gly Tyr Ile Asp Tyr Ala Glu Phe Ala Lys Ser Leu Gin

[0138] (amino acid sequence 106-119 in SEQ ID No.4)

[0139] and immunized rabbits as an immunogen to obtain serum aftermeasuring levels of antibody to the protein. Anti-SDNSF polyclonalantibodies were purified using affinity column, in which each peptidefragment used as an immunogen was bound.

EXAMPLE 8 Investigation of Secretory Pathway of SDNSF

[0140] Modified SDNSF proteins, which were tagged with inserted FLAG atN terminus (FLAG-SDNSF) or with FLAG-6His at C terminus(SDNSF—C′FLAG-6His) of rat SDNSF, were expressed in 293T cells. Thesecretion of these tagged SDNSF proteins into culture supernatant wasexamined with Western blot analysis using both anti-SDNSF and anti-FLAGantibodies (FIG. 3).

EXAMPLE 9 Localization of Rat SDNSF

[0141] Total RNA was extracted from adult rat brain, heart, lung, liver,spleen, kidney, testis, skeletal muscle and thymus with TRI_(zol)reagent™ (purchased from Life Technologies, Inc), and poly(A)⁺ RNA waspurified with Oligotex-dT30<Super>™ (purchased from Roche).

[0142] The poly(A)⁺ RNA from various tissues were subjected toformaldehyde-gel electrophoresis and blotting according to the method ofSambrook et al. (Molecular Cloning (1989)). The detection usinggel-purified and ³²P-labelled cDNA fragment as a probe revealed thatSDNSF were expressed in all tissues tested as shown in FIG. 4.

[0143] Total RNA was extracted from rat whole embryo and embryonicbrain, brain up to postnatal day 7, primary neurons and cell lines withTRI_(zol) reagent™ (purchased from Life Technologies, Inc), and mRNAexpression were examined by using RT-PCR. As shown in FIG. 5, the SDNSFtranscript was expressed in cultured stem cells, primary neurons andneural stem cells. The SDNSF transcript was also expressed in humanglioblastoma UG251 cells and mouse neuroblastoma N18 cells, but not inprimary glia cells and rat glioma C6 cells. Furthermore, the expressionof SDNSF protein was detected in human glioblastoma UG251 cells andmouse neuroblastoma N18 cells by Western blot analysis (FIG. 6).

EXAMPLE 10 Viability Assays of SDNSF on Neurons and Stem Cells

[0144] Based on the localization of SDNSF, biological effects of SDNSFon neurons and stem cells were examined.

[0145] Modified SDNSF protein (SDNSF-FLAG-6His), which was purified byusing Ni-NTA method utilizing His structure in the molecule, was addedto the cultures of rat primary hippocampal neurons and neural stem cellsderived from rat hippocampus, and WST reduction assay was performed tomeasure the number of viable cell at day 4. As shown in FIG. 7, inprimary neurons, SDNSF at the concentration of 100 ng/ml was effectiveon cell survival as compared with control group. In neural stem cells,it was shown that SDNSF tended to improve viable cell numbers ascompared with control group. It was also shown that SDNSF improved theviability of neural stem cells derived from rat hippocampus in adose-dependent manner when cells were cultured in FGF-2 (fibroblastgrowth factor-2) minus growth medium for 5 days (FIGS. 8 and 9).

EXAMPLE 11 Test for SDNSF's Effect on Self-Renewal and Differentiationof Neural Stem Cells

[0146] After neural stem cells derived from rat hippocampus werecultured in the SDNSF⁺/FGF-2⁻ medium for 5 days, cells were replated onnoncoated plates and grown for 6 days in growth medium containing FGF-2(20 ng/ml), and the number of neurospheres were counted. Aneuronalmarker, Tuj-1-possitive cells were also counted. AS shown in FIGS. 10and 11, the number of neurospheres/well from SDNSF-treated group wassignificantly larger than that from the control group. Proportion ofneurons in the neurospheres from SDNSF-treated group was significantlylarger than that from the control group. These results indicated thatSDNSF had activities for retaining self-renewal potentials of neuralstem cells and for promoting the differentiation of neural stem cellsinto neuronal and glial phenotypes as well as FGF-2.

1 20 1 1771 DNA Rattus rattus 1 gcgtcagggg gacgcagctg gcaaggttcatccacaagtg cttcgcgact gcgtcaggga 60 ttatcagggt actggaagca tggcatccctgcagctgctc agaggtccct tcctgtgtgt 120 tctgctctgg gccttttgtg ttcctggtgccagggcccag gagcatgggg ctggtgtcca 180 ccatggcagc gtgggcctgg acaagagcacagtgcacgac caagagcaca ttatggaaca 240 tctggaaggt gtcatcaacc agccagaggcggagatgtcc ccacaggaac tacagctcca 300 ttatttcaaa atgcatgatt acgatggcaacagtttgctt gatggcttag agctctcgac 360 ggccatcact cacgtgcaca aggaggaggggagtgagcag gtcccaccca tgagcgagga 420 cgagctcatc agcatcatag acggtgtcctgagagacgac gacaagaaca atgacggcta 480 catcgactat gcagagtttg ccaagtcgctgcagtaggcg gcaggccctt tcctgtatgc 540 acacgtgacc cttgctaatg tgatggacattctggtaatg agaagcagct tatttctgtc 600 tactgctgca gcgctggtaa agcctgtggcagtctgttag actggggtag gaggaagcca 660 caaggaatac ggagagaagt ggggcagtgtcaatgtgtgt ttaaacctgt tggacaagag 720 ctcgaacctt ccgaagggtg gtggggtatctcaagctccc gggaacctga ctctagatgc 780 cactctaact tcttgatgtt atttcatgctacctgaaaag taaagacagt ctgctttgcc 840 aagtggagac ttcagtgacg gtggagggagagccaaaagc cgcgtatctt cccagttggg 900 tcctgctctg ggcagatgtg gtcagtatgctgttccccag gcatacagca tcacgtccta 960 aagccacagc aggagaagaa tgtcacccacggagtccacc agacacagag tgaagactcc 1020 ttacccactg gcattttgga agcgaagcaccactggcctg aatacttagc cttttcagat 1080 cttcagtttc cttcacaact actgccacaccctgtgctct gtcatttcag cccgagagaa 1140 accttgaatt gggtgtgctc tccgctcaccacccaccgtt tgagctccct gaccttgtgt 1200 tttatccttg ctcccagggc tcccttcttggcttatgaac tattaacttg gtatcgcagg 1260 tttaaactgt cagctgctct agcctaagtcagaccagaaa agatcagtca ttaagggtgg 1320 tggctaacct tatccaagtt ttgaaggaatgtttttaaaa ttacctcttt gagcctgaat 1380 atgataattc ttttaatttc agggaagaacagaaaaggaa gagcagtagt agctgaaaga 1440 gaaacagcca taggtcgtac tttgcgttgtgaaacgtcat agacttactg taaacgaatc 1500 cagaatgatg gtgggatcag aaaaagaaactgaatcaaat ttgctttacg atgtatagag 1560 acttattttc tttattaaag tattcttgtaagaaaactta cgtatttgta aaacagtttt 1620 ctgtgtcaag tatttgtgca atcggagctgacttgtaaac tattcttgta agatctcatt 1680 attttgaaag atttatataa tgaactctgactatctgaca ataaaatgga tgaaaaagta 1740 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a1771 2 435 DNA Rattus rattus 2 atggcatccc tgcagctgct cagaggtcccttcctgtgtg ttctgctctg ggccttttgt 60 gttcctggtg ccagggccca ggagcatggggctggtgtcc accatggcag cgtgggcctg 120 gacaagagca cagtgcacga ccaagagcacattatggaac atctggaagg tgtcatcaac 180 cagccagagg cggagatgtc cccacaggaactacagctcc attatttcaa aatgcatgat 240 tacgatggca acagtttgct tgatggcttagagctctcga cggccatcac tcacgtgcac 300 aaggaggagg ggagtgagca ggtcccacccatgagcgagg acgagctcat cagcatcata 360 gacggtgtcc tgagagacga cgacaagaacaatgacggct acatcgacta tgcagagttt 420 gccaagtcgc tgcag 435 3 1771 DNARattus rattus CDS (80)..(514) sig_peptide (80)..(157) mat_peptide(158)..(514) 3 gcgtcagggg gacgcagctg gcaaggttca tccacaagtg cttcgcgactgcgtcaggga 60 ttatcagggt actggaagc atg gca tcc ctg cag ctg ctc aga ggtccc ttc 112 Met Ala Ser Leu Gln Leu Leu Arg Gly Pro Phe -25 -20 ctg tgtgtt ctg ctc tgg gcc ttt tgt gtt cct ggt gcc agg gcc cag 160 Leu Cys ValLeu Leu Trp Ala Phe Cys Val Pro Gly Ala Arg Ala Gln -15 -10 -5 -1 1 gagcat ggg gct ggt gtc cac cat ggc agc gtg ggc ctg gac aag agc 208 Glu HisGly Ala Gly Val His His Gly Ser Val Gly Leu Asp Lys Ser 5 10 15 aca gtgcac gac caa gag cac att atg gaa cat ctg gaa ggt gtc atc 256 Thr Val HisAsp Gln Glu His Ile Met Glu His Leu Glu Gly Val Ile 20 25 30 aac cag ccagag gcg gag atg tcc cca cag gaa cta cag ctc cat tat 304 Asn Gln Pro GluAla Glu Met Ser Pro Gln Glu Leu Gln Leu His Tyr 35 40 45 ttc aaa atg catgat tac gat ggc aac agt ttg ctt gat ggc tta gag 352 Phe Lys Met His AspTyr Asp Gly Asn Ser Leu Leu Asp Gly Leu Glu 50 55 60 65 ctc tcg acg gccatc act cac gtg cac aag gag gag ggg agt gag cag 400 Leu Ser Thr Ala IleThr His Val His Lys Glu Glu Gly Ser Glu Gln 70 75 80 gtc cca ccc atg agcgag gac gag ctc atc agc atc ata gac ggt gtc 448 Val Pro Pro Met Ser GluAsp Glu Leu Ile Ser Ile Ile Asp Gly Val 85 90 95 ctg aga gac gac gac aagaac aat gac ggc tac atc gac tat gca gag 496 Leu Arg Asp Asp Asp Lys AsnAsn Asp Gly Tyr Ile Asp Tyr Ala Glu 100 105 110 ttt gcc aag tcg ctg cagtaggcggcag gccctttcct gtatgcacac 544 Phe Ala Lys Ser Leu Gln 115gtgacccttg ctaatgtgat ggacattctg gtaatgagaa gcagcttatt tctgtctact 604gctgcagcgc tggtaaagcc tgtggcagtc tgttagactg gggtaggagg aagccacaag 664gaatacggag agaagtgggg cagtgtcaat gtgtgtttaa acctgttgga caagagctcg 724aaccttccga agggtggtgg ggtatctcaa gctcccggga acctgactct agatgccact 784ctaacttctt gatgttattt catgctacct gaaaagtaaa gacagtctgc tttgccaagt 844ggagacttca gtgacggtgg agggagagcc aaaagccgcg tatcttccca gttgggtcct 904gctctgggca gatgtggtca gtatgctgtt ccccaggcat acagcatcac gtcctaaagc 964cacagcagga gaagaatgtc acccacggag tccaccagac acagagtgaa gactccttac 1024ccactggcat tttggaagcg aagcaccact ggcctgaata cttagccttt tcagatcttc 1084agtttccttc acaactactg ccacaccctg tgctctgtca tttcagcccg agagaaacct 1144tgaattgggt gtgctctccg ctcaccaccc accgtttgag ctccctgacc ttgtgtttta 1204tccttgctcc cagggctccc ttcttggctt atgaactatt aacttggtat cgcaggttta 1264aactgtcagc tgctctagcc taagtcagac cagaaaagat cagtcattaa gggtggtggc 1324taaccttatc caagttttga aggaatgttt ttaaaattac ctctttgagc ctgaatatga 1384taattctttt aatttcaggg aagaacagaa aaggaagagc agtagtagct gaaagagaaa 1444cagccatagg tcgtactttg cgttgtgaaa cgtcatagac ttactgtaaa cgaatccaga 1504atgatggtgg gatcagaaaa agaaactgaa tcaaatttgc tttacgatgt atagagactt 1564attttcttta ttaaagtatt cttgtaagaa aacttacgta tttgtaaaac agttttctgt 1624gtcaagtatt tgtgcaatcg gagctgactt gtaaactatt cttgtaagat ctcattattt 1684tgaaagattt atataatgaa ctctgactat ctgacaataa aatggatgaa aaagtaaaaa 1744aaaaaaaaaa aaaaaaaaaa aaaaaaa 1771 4 145 PRT Rattus rattus 4 Met Ala SerLeu Gln Leu Leu Arg Gly Pro Phe Leu Cys Val Leu Leu -25 -20 -15 Trp AlaPhe Cys Val Pro Gly Ala Arg Ala Gln Glu His Gly Ala Gly -10 -5 -1 1 5Val His His Gly Ser Val Gly Leu Asp Lys Ser Thr Val His Asp Gln 10 15 20Glu His Ile Met Glu His Leu Glu Gly Val Ile Asn Gln Pro Glu Ala 25 30 35Glu Met Ser Pro Gln Glu Leu Gln Leu His Tyr Phe Lys Met His Asp 40 45 50Tyr Asp Gly Asn Ser Leu Leu Asp Gly Leu Glu Leu Ser Thr Ala Ile 55 60 6570 Thr His Val His Lys Glu Glu Gly Ser Glu Gln Val Pro Pro Met Ser 75 8085 Glu Asp Glu Leu Ile Ser Ile Ile Asp Gly Val Leu Arg Asp Asp Asp 90 95100 Lys Asn Asn Asp Gly Tyr Ile Asp Tyr Ala Glu Phe Ala Lys Ser Leu 105110 115 Gln 5 823 DNA Homo sapiens 5 tggtgaggcc cgaggcgttg gagggcttcgcgtctgcttc ggagaccgta aggatattga 60 tgaccatgag atccctgctc agaacccccttcctgtgtgg cctgctctgg gccttttgtg 120 ccccaggcgc cagggctgag gagcctgcagccagcttctc ccaacccggc agcatgggcc 180 tggataagaa cacagtgcac gaccaagagcatatcatgga gcatctagaa ggtgtcatca 240 acaaaccaga ggcggagatg tcgccacaagaattgcagct ccattacttc aaaatgcatg 300 attatgatgg caataatttg cttgatggcttagaactctc cacagccatc actcatgtcc 360 ataaggagga agggagtgaa caggcaccactaatgagtga agatgaactg attaacataa 420 tagatggtgt tttgagagat gatgacaagaacaatgatgg atacattgac tatgctgaat 480 ttgcaaaatc actgcagtag atgttatttggccatctcct ggttatatac aaatgtgacc 540 cgtgataatg tgattgaaca ctttagtaatgcaaaataac tcatttccaa ctactgctgc 600 agcattttgg taaaaacctg tagcgattcgttacactggg gtgagaagag ataagagaaa 660 tgaaagagaa gagaaatggg acatctaatagtccctaagt gctattaaat accttattgg 720 acaaggaaaa acaacaaaaa aaaatattagtctgtattaa tgctgctgat aaagacgtac 780 ccaagactgg gaagaaaaaa aaaaaaaaaaaaaaaaaaaa aaa 823 6 438 DNA Homo sapiens 6 atgaccatga gatccctgctcagaaccccc ttcctgtgtg gcctgctctg ggccttttgt 60 gccccaggcg ccagggctgaggagcctgca gccagcttct cccaacccgg cagcatgggc 120 ctggataaga acacagtgcacgaccaagag catatcatgg agcatctaga aggtgtcatc 180 aacaaaccag aggcggagatgtcgccacaa gaattgcagc tccattactt caaaatgcat 240 gattatgatg gcaataatttgcttgatggc ttagaactct ccacagccat cactcatgtc 300 cataaggagg aagggagtgaacaggcacca ctaatgagtg aagatgaact gattaacata 360 atagatggtg ttttgagagatgatgacaag aacaatgatg gatacattga ctatgctgaa 420 tttgcaaaat cactgcag 4387 823 DNA Homo sapiens CDS (60)..(497) sig_peptide (60)..(137)mat_peptide (138)..(497) 7 tggtgaggcc cgaggcgttg gagggcttcg cgtctgcttcggagaccgta aggatattg 59 atg acc atg aga tcc ctg ctc aga acc ccc ttc ctgtgt ggc ctg ctc 107 Met Thr Met Arg Ser Leu Leu Arg Thr Pro Phe Leu CysGly Leu Leu -25 -20 -15 tgg gcc ttt tgt gcc cca ggc gcc agg gct gag gagcct gca gcc agc 155 Trp Ala Phe Cys Ala Pro Gly Ala Arg Ala Glu Glu ProAla Ala Ser -10 -5 -1 1 5 ttc tcc caa ccc ggc agc atg ggc ctg gat aagaac aca gtg cac gac 203 Phe Ser Gln Pro Gly Ser Met Gly Leu Asp Lys AsnThr Val His Asp 10 15 20 caa gag cat atc atg gag cat cta gaa ggt gtc atcaac aaa cca gag 251 Gln Glu His Ile Met Glu His Leu Glu Gly Val Ile AsnLys Pro Glu 25 30 35 gcg gag atg tcg cca caa gaa ttg cag ctc cat tac ttcaaa atg cat 299 Ala Glu Met Ser Pro Gln Glu Leu Gln Leu His Tyr Phe LysMet His 40 45 50 gat tat gat ggc aat aat ttg ctt gat ggc tta gaa ctc tccaca gcc 347 Asp Tyr Asp Gly Asn Asn Leu Leu Asp Gly Leu Glu Leu Ser ThrAla 55 60 65 70 atc act cat gtc cat aag gag gaa ggg agt gaa cag gca ccacta atg 395 Ile Thr His Val His Lys Glu Glu Gly Ser Glu Gln Ala Pro LeuMet 75 80 85 agt gaa gat gaa ctg att aac ata ata gat ggt gtt ttg aga gatgat 443 Ser Glu Asp Glu Leu Ile Asn Ile Ile Asp Gly Val Leu Arg Asp Asp90 95 100 gac aag aac aat gat gga tac att gac tat gct gaa ttt gca aaatca 491 Asp Lys Asn Asn Asp Gly Tyr Ile Asp Tyr Ala Glu Phe Ala Lys Ser105 110 115 ctg cag tagatgttat ttggccatct cctggttata tacaaatgtgacccgtgata 547 Leu Gln 120 atgtgattga acactttagt aatgcaaaat aactcatttccaactactgc tgcagcattt 607 tggtaaaaac ctgtagcgat tcgttacact ggggtgagaagagataagag aaatgaaaga 667 gaagagaaat gggacatcta atagtcccta agtgctattaaataccttat tggacaagga 727 aaaacaacaa aaaaaaatat tagtctgtat taatgctgctgataaagacg tacccaagac 787 tgggaagaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 823 8146 PRT Homo sapiens 8 Met Thr Met Arg Ser Leu Leu Arg Thr Pro Phe LeuCys Gly Leu Leu -25 -20 -15 Trp Ala Phe Cys Ala Pro Gly Ala Arg Ala GluGlu Pro Ala Ala Ser -10 -5 -1 1 5 Phe Ser Gln Pro Gly Ser Met Gly LeuAsp Lys Asn Thr Val His Asp 10 15 20 Gln Glu His Ile Met Glu His Leu GluGly Val Ile Asn Lys Pro Glu 25 30 35 Ala Glu Met Ser Pro Gln Glu Leu GlnLeu His Tyr Phe Lys Met His 40 45 50 Asp Tyr Asp Gly Asn Asn Leu Leu AspGly Leu Glu Leu Ser Thr Ala 55 60 65 70 Ile Thr His Val His Lys Glu GluGly Ser Glu Gln Ala Pro Leu Met 75 80 85 Ser Glu Asp Glu Leu Ile Asn IleIle Asp Gly Val Leu Arg Asp Asp 90 95 100 Asp Lys Asn Asn Asp Gly TyrIle Asp Tyr Ala Glu Phe Ala Lys Ser 105 110 115 Leu Gln 120 9 1815 DNAMus musculus 9 gtgcggagaa aagcgtccca gggacggcag ctggcaaggt tcacgttggagtgcttcgcg 60 actgcgtcgg ggattatcgg ggtacccacc cggaagcatg gcaaccctacagctgctcag 120 agctcccttg ctgtgtgtcc tgctttgggt cttttgtgct ccaggtgccagagcccatga 180 ccatggggct gatgtccatc atggcagcgt gggcctggat aagagcacagtgcacgacca 240 agagcacatc atggaacatc tggaaggtgt catcgaccag ccagaggcggagatgtcccc 300 acaggaactg cagctccatt acttcaaaat gcatgattac gacggcaacagtttgcttga 360 cggcctagag ctctccatag ccatcactca cgtgcacaag gaggaggggagtgagcaggc 420 gccagtcatg agcgaggatg agctcgtcag catcatagat ggtgtcctgagggacgatga 480 caagaacaat gacggctaca tcgactacgc tgagtttgca aagtcactgcagtagaccgt 540 tggctctttc ctttgtgcac atgtgaccct tgctaatgtg atggacgtgtctggtaatgc 600 gaaacaactt atttccgtct actgctcagc actttggtaa gagcctgtggcagtctgtaa 660 gagtggggtg aggaagaagc cacatgactg tggagagaag tgggacaggcctcagtccct 720 agaggtgtgt ttaagcttgt tgggcaagag ccggatgcgg atcttcggaagggcggtggg 780 tatcccgagt tctcaggaat ccgactgtag aatgccactc tgacttcttgatgttaatcc 840 atgctaccta aagtaaagac aggctgcttg gccaagtgga cacacttgagaaacagtgga 900 gggagagtgt gaaagccaca cgcttgccct ggttggtcct gtctttaggcagatgtggtc 960 agtattctgt tccccaggca tacagcatca tatattaaag ccacagcagaagaggaatgt 1020 cgcccactga ggccacccag atgcagagtc taggattcct tgcccactggccttttggaa 1080 atgaagcacc actggcctga ataattagca ttttccagat cttcagtatcttccacaact 1140 actgccatac cctgtgttgt atcatttgac caggagggaa accttgaattggggtgtgtt 1200 ctctaatcac tttccactgt ctgagctttc ctgacccctg tattgtatccttgctcccag 1260 ggctcccttc atggcttgtg aactgttaac ttggtatctc aggttaaactgtcagctggt 1320 ctagcctgag cgaggcctga gaccatcagt cactaagagc agtggctaacctcatcgaag 1380 ttggaaggaa tgtttttaaa attacctctt cgagcctgaa tacaaagaataaaagaataa 1440 aagaattctt ttaatttcag ggaagatcag aaaagaaagc ctaaagccctttagcgttgt 1500 gaacctcagt agtagctgaa agagaagctg ccacaggttg tacttgctctgtgagatgtt 1560 gtagacattc cgtaagagaa tccagaatga tagcaggatc aggaaagaaatggagccaaa 1620 tctgctctaa ggtgaataga gacttatttt tctttattaa agtattcttgtaagacagtt 1680 ttctgtgtca agtatttgtg aaatcagagc tgacatgtaa gctattcttgtaatatctca 1740 ttattttgaa agatttatat aatgaactct ggctatctga caataaaatggatgaaaaag 1800 caaaaaaaaa aaaaa 1815 10 435 DNA Mus musculus 10atggcaaccc tacagctgct cagagctccc ttgctgtgtg tcctgctttg ggtcttttgt 60gctccaggtg ccagagccca tgaccatggg gctgatgtcc atcatggcag cgtgggcctg 120gataagagca cagtgcacga ccaagagcac atcatggaac atctggaagg tgtcatcgac 180cagccagagg cggagatgtc cccacaggaa ctgcagctcc attacttcaa aatgcatgat 240tacgacggca acagtttgct tgacggccta gagctctcca tagccatcac tcacgtgcac 300aaggaggagg ggagtgagca ggcgccagtc atgagcgagg atgagctcgt cagcatcata 360gatggtgtcc tgagggacga tgacaagaac aatgacggct acatcgacta cgctgagttt 420gcaaagtcac tgcag 435 11 1815 DNA Mus musculus CDS (98)..(532)sig_peptide (98)..(175) mat_peptide (176)..(532) 11 gtgcggagaaaagcgtccca gggacggcag ctggcaaggt tcacgttgga gtgcttcgcg 60 actgcgtcggggattatcgg ggtacccacc cggaagc atg gca acc cta cag ctg 115 Met Ala ThrLeu Gln Leu -25 ctc aga gct ccc ttg ctg tgt gtc ctg ctt tgg gtc ttt tgtgct cca 163 Leu Arg Ala Pro Leu Leu Cys Val Leu Leu Trp Val Phe Cys AlaPro -20 -15 -10 -5 ggt gcc aga gcc cat gac cat ggg gct gat gtc cat catggc agc gtg 211 Gly Ala Arg Ala His Asp His Gly Ala Asp Val His His GlySer Val -1 1 5 10 ggc ctg gat aag agc aca gtg cac gac caa gag cac atcatg gaa cat 259 Gly Leu Asp Lys Ser Thr Val His Asp Gln Glu His Ile MetGlu His 15 20 25 ctg gaa ggt gtc atc gac cag cca gag gcg gag atg tcc ccacag gaa 307 Leu Glu Gly Val Ile Asp Gln Pro Glu Ala Glu Met Ser Pro GlnGlu 30 35 40 ctg cag ctc cat tac ttc aaa atg cat gat tac gac ggc aac agtttg 355 Leu Gln Leu His Tyr Phe Lys Met His Asp Tyr Asp Gly Asn Ser Leu45 50 55 60 ctt gac ggc cta gag ctc tcc ata gcc atc act cac gtg cac aaggag 403 Leu Asp Gly Leu Glu Leu Ser Ile Ala Ile Thr His Val His Lys Glu65 70 75 gag ggg agt gag cag gcg cca gtc atg agc gag gat gag ctc gtc agc451 Glu Gly Ser Glu Gln Ala Pro Val Met Ser Glu Asp Glu Leu Val Ser 8085 90 atc ata gat ggt gtc ctg agg gac gat gac aag aac aat gac ggc tac499 Ile Ile Asp Gly Val Leu Arg Asp Asp Asp Lys Asn Asn Asp Gly Tyr 95100 105 atc gac tac gct gag ttt gca aag tca ctg cag tagaccgttggctctttcct 552 Ile Asp Tyr Ala Glu Phe Ala Lys Ser Leu Gln 110 115ttgtgcacat gtgacccttg ctaatgtgat ggacgtgtct ggtaatgcga aacaacttat 612ttccgtctac tgctcagcac tttggtaaga gcctgtggca gtctgtaaga gtggggtgag 672gaagaagcca catgactgtg gagagaagtg ggacaggcct cagtccctag aggtgtgttt 732aagcttgttg ggcaagagcc ggatgcggat cttcggaagg gcggtgggta tcccgagttc 792tcaggaatcc gactgtagaa tgccactctg acttcttgat gttaatccat gctacctaaa 852gtaaagacag gctgcttggc caagtggaca cacttgagaa acagtggagg gagagtgtga 912aagccacacg cttgccctgg ttggtcctgt ctttaggcag atgtggtcag tattctgttc 972cccaggcata cagcatcata tattaaagcc acagcagaag aggaatgtcg cccactgagg 1032ccacccagat gcagagtcta ggattccttg cccactggcc ttttggaaat gaagcaccac 1092tggcctgaat aattagcatt ttccagatct tcagtatctt ccacaactac tgccataccc 1152tgtgttgtat catttgacca ggagggaaac cttgaattgg ggtgtgttct ctaatcactt 1212tccactgtct gagctttcct gacccctgta ttgtatcctt gctcccaggg ctcccttcat 1272ggcttgtgaa ctgttaactt ggtatctcag gttaaactgt cagctggtct agcctgagcg 1332aggcctgaga ccatcagtca ctaagagcag tggctaacct catcgaagtt ggaaggaatg 1392tttttaaaat tacctcttcg agcctgaata caaagaataa aagaataaaa gaattctttt 1452aatttcaggg aagatcagaa aagaaagcct aaagcccttt agcgttgtga acctcagtag 1512tagctgaaag agaagctgcc acaggttgta cttgctctgt gagatgttgt agacattccg 1572taagagaatc cagaatgata gcaggatcag gaaagaaatg gagccaaatc tgctctaagg 1632tgaatagaga cttatttttc tttattaaag tattcttgta agacagtttt ctgtgtcaag 1692tatttgtgaa atcagagctg acatgtaagc tattcttgta atatctcatt attttgaaag 1752atttatataa tgaactctgg ctatctgaca ataaaatgga tgaaaaagca aaaaaaaaaa 1812aaa 1815 12 145 PRT Mus musculus 12 Met Ala Thr Leu Gln Leu Leu Arg AlaPro Leu Leu Cys Val Leu Leu -25 -20 -15 Trp Val Phe Cys Ala Pro Gly AlaArg Ala His Asp His Gly Ala Asp -10 -5 -1 1 5 Val His His Gly Ser ValGly Leu Asp Lys Ser Thr Val His Asp Gln 10 15 20 Glu His Ile Met Glu HisLeu Glu Gly Val Ile Asp Gln Pro Glu Ala 25 30 35 Glu Met Ser Pro Gln GluLeu Gln Leu His Tyr Phe Lys Met His Asp 40 45 50 Tyr Asp Gly Asn Ser LeuLeu Asp Gly Leu Glu Leu Ser Ile Ala Ile 55 60 65 70 Thr His Val His LysGlu Glu Gly Ser Glu Gln Ala Pro Val Met Ser 75 80 85 Glu Asp Glu Leu ValSer Ile Ile Asp Gly Val Leu Arg Asp Asp Asp 90 95 100 Lys Asn Asn AspGly Tyr Ile Asp Tyr Ala Glu Phe Ala Lys Ser Leu 105 110 115 Gln 13 38DNA Rattus rattus misc_feature (30)..(30) n = a, c, g or t 13 tcccgattgaattctagacc tgcctcgagn nnnnnnnn 38 14 186 PRT Caenorhabditis elegans 14Met Ala Ala Asn Ile Leu Val Val Ser Cys Leu Ile Leu Gly Ser Phe 1 5 1015 Ala His Gln Pro Gln Gln Phe Pro Gly Ser Asn Gln Gln Gln Pro Gln 20 2530 Gln Gly Gly Gln Ala Glu Gln Ala Gln His Ala Gln Pro Gly Gln Gln 35 4045 Gln Phe Gly Gly Glu Gln Ala Arg Asp Glu His His Ile Lys Glu His 50 5560 Leu Asp Gly Lys Val Asp Pro Thr Ala Asn Met Thr Pro Glu Gln Leu 65 7075 80 Pro Phe His Tyr Phe Asn Met His Asp Leu Asp Lys Asn Gly Lys Leu 8590 95 Asp Gly Val Glu Leu Ile Lys Ala Ile Thr His Phe His Ala Glu Asn100 105 110 Pro Gly Pro Gln His Thr Gln Asn Asn Ala Asn Ala Asn His GlnPro 115 120 125 Pro Pro Leu Pro Ser Glu Val Glu Leu Glu Thr Met Ile AspSer Ile 130 135 140 Leu Lys Asp Asp Asp Phe Asn Ala Asp Gly Phe Ile AspTyr Gly Glu 145 150 155 160 Phe Leu Lys Ala Gln Lys Leu Arg Glu Asp GlnAla Arg Ser His Gln 165 170 175 Glu Gln Met Gln Lys Ala Gly Gly Thr Gln180 185 15 168 PRT Drosophila melanogaster 15 Met Cys Asn Leu Ser AsnLeu Leu Asn Phe Ile Ile Cys Ile Ala Ser 1 5 10 15 Phe Ser Gln Asn PheAsp Ala Thr Leu Ala Val Lys Arg Gly Pro His 20 25 30 His Pro Arg Gly GluThr Arg Arg Val Asp Gln His Leu Thr His Glu 35 40 45 Glu His Arg Ile AspAsp Asp Leu Lys Asp Met Gly Val Gln Ala Asn 50 55 60 Leu Asp Asp Leu SerGlu Glu Glu Lys Ile Phe Tyr Met Phe Lys Ala 65 70 75 80 His Asp Asn AspAsn Asn Asn Ala Leu Asp Gly Leu Glu Met Ile Gln 85 90 95 Ser Ala Met HisHis Asn Tyr Asp Tyr Phe Lys Asn Asn Glu Arg Asp 100 105 110 Ala Tyr LeuGln Asn Ala Thr Asp Glu Leu Glu His Phe Ile Glu Ala 115 120 125 Ile AspLys Phe Leu Leu Ile Ala Asp Asp Asn Asn Asp Gly Leu Leu 130 135 140 HisTyr Pro Glu Phe Val Lys Ala Ile Thr Gly Gly Lys Glu Gln Pro 145 150 155160 Asn Val Asp Arg Asn Ile Leu Arg 165 16 29 PRT Rattus rattus 16 GluPhe Lys Glu Ala Phe Ala Leu Phe Asp Lys Asp Gly Asp Gly Thr 1 5 10 15Ile Thr Thr Lys Glu Leu Gly Thr Val Met Arg Ser Leu 20 25 17 29 PRTRattus rattus 17 Glu Leu Gln Asp Met Ile Asn Glu Val Asp Ala Asp Gly AsnGly Thr 1 5 10 15 Ile Asp Phe Pro Glu Phe Leu Ser Leu Met Ala Arg Lys 2025 18 29 PRT Rattus rattus 18 Glu Leu Ile Glu Ala Phe Lys Val Phe AspArg Asp Gly Asn Gly Leu 1 5 10 15 Ile Ser Ala Ala Glu Leu Arg His ValMet Thr Asn Leu 20 25 19 29 PRT Rattus rattus 19 Glu Val Asp Glu Met IleArg Glu Ala Asp Ile Asp Gly Asp Gly His 1 5 10 15 Ile Asn Tyr Glu GluPhe Val Arg Met Met Val Ala Lys 20 25 20 12 PRT Artificial Sequence Loopconsensus sequence 20 Asp Xaa Asp Gly Asp Gly Xaa Ile Asp Xaa Xaa Glu 15 10

1. A substantially purified form of a polypeptide comprising the aminoacid sequence shown in SEQ ID NO. 4, 8 or 12, a homologue thereof, afragment thereof or a homologue of the fragment.
 2. The polypeptideaccording to claim 1 that comprises the amino acid sequence shown in SEQID NO. 4, 8 or
 12. 3. A cDNA encoding the polypeptide according toclaim
 1. 4. The cDNA according to claim 3, comprising the nucleotidesequence shown in SEQ ID NO. 1, 2, 5, 6, 9 or 10, or a fragmentselectively hybridized to the sequence.
 5. A replication or expressionvector comprising the cDNA according to claim
 3. 6. A host celltransformed with the replication or expression vector according to claim5.
 7. A process for producing a substantially purified form of apolypeptide comprising the amino acid sequence shown in SEQ ID NO. 4, 8or 12, a homologue thereof, a fragment thereof or a homologue of thefragment, which comprises culturing the host cell according to claim 6under a condition effective to express the polypeptide.
 8. A monoclonalor polyclonal antibody against the polypeptide according to claim
 1. 9.A pharmaceutical composition containing the polypeptide according toclaim 1 or the antibody according to claim 8, in association with apharmaceutically acceptable excipient and/or carrier.
 10. Apharmaceutical composition effective for the medical treatment of aneurodegenerative disease, containing the polypeptide according to claim1 or the antibody according to claim 8, in association with apharmaceutically acceptable excipient and/or carrier.
 11. Thepharmaceutical composition according to claim 10, in which theneurodegenerative disease is an injury to the central nerve system. 12.A method for measuring the polypeptide according to claim
 1. 13. Animmunochemical method for measuring a substantially purified form of apolypeptide comprising the amino acid sequence shown in SEQ ID NO. 4, 8or 12, a homologue thereof, a fragment thereof or a homologue of thefragment, comprising using the antibody according to claim
 8. 14. Areagent for detecting a substantially purified form of a polypeptidecomprising the amino acid sequence shown in SEQ ID NO. 4, 8 or 12, ahomologue thereof, a fragment thereof or a homologue of the fragment,comprising using the method according to claim 12 or
 13. 15. A reagentfor testing tumor, comprising using the method according to claim 12 or13.
 16. The reagent according to claim 14, in which the tumor is a braintumor.
 17. A method for screening a compound having agonistic orantagonistic activity against a polypeptide, comprising using thepolypeptide according to claim
 1. 18. An agent for the treatment of aninjury to the central nerve system which comprises, as an activeingredient, the polypeptide according to claim
 1. 19. The reagentaccording to claim 18, in which the injury to the central nerve systemis the one caused by a brain infarction.
 20. The reagent according toclaim 18, in which the injury to the central nerve system is the onecaused by a brain hemorrhage.
 21. The reagent according to claim 18, inwhich the injury to the central nerve system is the one caused by aspinal cord injury.
 22. A method for treating a neurodegenerativedisease, which comprises administering the polypeptide according toclaim 1 or the antibody according to claim
 8. 23. The method accordingto claim 22, in which the neurodegenerative disease is an injury to thecentral nerve system.
 24. A method for treating an injury to the centralnerve system, which comprises administering the polypeptide according toclaim
 1. 25. The method according to claim 24, in which the injury tothe central nerve system is the one caused by a brain infarction. 26.The method according to claim 24, in which the injury to the centralnerve system is the one caused by a brain hemorrhage.
 27. The methodaccording to claim 24, in which the injury to the central nerve systemis the one caused by a spinal cord injury.